A NOVEL EXOCYTOPLASMIC ENDONUCLEASE FROM STREPTOMYCES-ANTIBIOTICUS

A new exocytoplasmic, nutritionally controlled endodeoxyribonuclease ( EC 3.1.21.-) was purified to homogeneity from Streptomyces antibioticu s. The enzyme showed an apparent molecular mass of 29 kDa (being activ e in the monomeric form) and a pI of similar to 7.8. The nuclease hydr olysed endonucleolytically double-stranded circular and linear DNA. Th e enzyme makes nicks in one strand of the DNA in G-rich regions, leavi ng either 5' or 3' short, single-stranded overhangs with 3'-hydroxy an d 5'-phosphate termini. Breaks in the DNA occur when two nicks in oppo site strands are close together. The enzyme had an optimum pH of 7.5 a nd an absolute requirement for bivalent cations and greater than or eq ual to 100 mM NaCl in the reaction buffer. Activity was greatly dimini shed in the presence of phosphate, Hg2+ or iodoacetate and was stimula ted by dimethyl sulphoxide. Single-stranded DNA was a much poorer subs trate than double-stranded DNA. The nuclease hydrolyses sequences of t hree or preferably more (dG).(dC) tracts in the DNA. The initial speci ficity shifts to other sequences (including sequences shorter than tho se initially hydrolysed) during the course of the reaction, giving the changing pattern of bands observed in agarose gels. 5-Methylcytosine- hemimethylated DNA is not hydrolysed by the nuclease. The properties o f this novel enzyme suggest a relationship with class II restriction e ndonucleases and also with some eukaryotic nucleases.